Interferon receptor 1 antibodies and their uses

ABSTRACT

The present invention provides isolated human monoclonal antibodies that bind to IFNAR-1 and that are capable of inhibiting the biological activity of Type I interferons. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for inhibiting Type I interferon-mediated disorders using the antibodies of the invention, including methods for treating autoimmune disorders, transplant rejection or Graft Versus Host Disease using the antibodies of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/890,494, filed on May 9, 2013, which is a divisional of U.S. patentapplication Ser. No. 12/704,948 and issued as U.S. Pat. No. 8,460,668,filed on Feb. 12, 2010, which is a divisional of U.S. patent applicationSer. No. 11/157,494, filed on Jun. 20, 2005 and issued as U.S. Pat. No.7,662,381, which claims priority to U.S. provisional application No.60/581,747, filed on Jun. 21, 2004, the contents of each of which arehereby incorporated by reference in their entirety herein, and to eachof which priority is claimed.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Sep. 9, 2016. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as077375.0962CONSEQ.txt, is 20,152 bytes and was created on Sep. 9, 2016.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

BACKGROUND OF THE INVENTION

Type I interferons (IFN) (IFN-α, IFN-β, IFN-ω, IFN-τ) are a family ofstructurally related cytokines having antiviral, antitumor andimmunomodulatory effects (Hardy et al. (2001) Blood 97:473; Cutrone andLanger (2001) J. Biol. Chem. 276:17140). The human IFNa locus includestwo subfamilies. The first subfamily consists of 14 non allelic genesand 4 pseudogenes having at least 80% homology. The second subfamily,all or omega (ω), contains 5 pseudogenes and 1 functional gene whichexhibits 70% homology with the IFNa genes (Weissmann and Weber (1986)Prog. Nucl. Acid Res. Mol. Biol., 33:251-300). The subtypes of IFNa havedifferent specific activities but they possess the same biologicalspectrum (Streuli et al. (1981) Proc. Natl. Acad. Sci. USA 78:2848) andhave the same cellular receptor (Agnet M. et al. in “Interferon 5” Ed.I. Gresser p. 1-22, Academic Press, London 1983).

The interferon β (IFN β) is encoded by a single gene which hasapproximately 50% homology with the IFNα genes.

Gamma interferon, which is produced by activated lymphocytes, does notpossess any homology with the alpha/beta interferons and it does notreact with their receptor.

All human type I interferons bind to a cell surface receptor (IFN alphareceptor, IFNAR) consisting of two transmembrane proteins, IFNAR-1 andIFNAR-2 (Uze et. al. (1990) Cell 60:225; Novick et al. (1994) Cell77:391). IFNAR-1 is essential for high affinity binding and differentialspecificity of the IFNAR complex (Cutrone, 2001, supra). Whilefunctional differences for each of the type I IFN subtypes have not beenidentified it is thought that each may exhibit different interactionswith the IFNAR receptor components leading to potentially diversesignaling outcomes (Cook et al. (1996) J. Biol. Chem. 271:13448). Inparticular, studies utilizing mutant forms of IFNAR1 and IFNAR2suggested that alpha and beta interferons signal differently through thereceptor by interacting differentially with respective chains (Lewerenzet al. (1998) J. Mol. Biol. 282:585).

Early functional studies of type I IFNs focused on innate defenseagainst viral infections (Haller et al. (1981) J. Exp. Med. 154:199;Lindenmann et al. (1981) Methods Enzymol. 78:181). More recent studies,however, implicate type I IFNs as potent immunoregulatory cytokines inthe adaptive immune response. Specifically, type I IFNs have been shownto facilitate differentiation of naive T cells along the Th1 pathway(Brinkmann et al. (1993) J. Exp. Med. 178:1655), to enhance antibodyproduction (Finkelman et al. (1991) J. Exp. Med. 174:1179) and tosupport the functional activity and survival of memory T cells (Santiniet al. (2000) J. Exp. Med. 191:1777; Tough et al. (1996) Science272:1947).

Recent work by a number of groups suggests that IFN-α may enhance thematuration or activation of dendritic cells (DCs) (Santini, et al.(2000) J. Exp. Med. 191:1777; Luft et al. (1998) J. Immunol. 161:1947;Luft et al. (2002) Int. Immunol. 14:367; Radvanyi et al. (1999) Scand.J. Immunol. 50:499). Furthermore, increased expression of type Iinterferons has been described in numerous autoimmune diseases (Fouliset al. (1987) Lancet 2:1423; Hooks et al. (1982) Arthritis Rheum.25:396; Hertzog et al. (1988) Clin. Immunol. Immunopathol. 48:192;Hopkins and Meager (1988) Clin. Exp. Immunol. 73:88; Arvin and Miller(1984) Arthritis Rheum. 27:582). The most studied examples of this areinsulin-dependent diabetes mellitus (IDDM) (Foulis (1987) supra) andsystemic lupus erythematosus (SLE) (Hooks (1982) supra), which areassociated with elevated levels of IFN-.alpha., and rheumatoid arthritis(RA) (Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller(1984), supra) in which IFN-β may play a more significant role.

Moreover, administration of interferon α has been reported to exacerbateunderlying disease in patients with psoriasis and multiple sclerosis andto induce an SLE like syndrome in patients without a previous history ofautoimmune disease. Interferon α has also been shown to induceglomerulonephritis in normal mice and to accelerate the onset of thespontaneous autoimmune disease of NZB/W mice. Further, IFN-α therapy hasbeen shown in some cases to lead to undesired side effects, includingfever and neurological disorders. Hence there are pathologicalsituations in which inhibition of Type I IFN activity may be beneficialto the patient and a need exists for agents effective in inhibiting TypeI IFN activity.

SUMMARY OF THE INVENTION

The present invention provides isolated human monoclonal antibodies thatbind to IFNAR-1 and inhibit the biological activity of type Iinterferon, preferably multiple type I interferons. Furthermore, theantibodies do not bind to the same epitope as the murine anti-IFNAR-1antibody, 64G12.

In one aspect, the invention pertains to an isolated human antibody, orantigen binding portion thereof, wherein the antibody specifically bindsto IFNAR-1 and exhibits one or more of the following properties:

-   -   a) binds to IFNAR-1 with a K_(D) of 1×10⁻⁷ M or greater        affinity;    -   b) inhibits the biological activity of multiple Type I        interferons;    -   c) inhibits the activity of IFN α 2b in a Daudi cell        proliferation assay;    -   d) inhibits the activity of IFN omega in a Daudi cell        proliferation assay;    -   e) inhibits IP-10 secretion by peripheral blood mononuclear        cells induced by IFN α 2b;    -   f) inhibits IP-10 secretion by peripheral blood mononuclear        cells induced by IFN omega;    -   g) inhibits dendritic cell development mediated by Systemic        Lupus Erythematosus plasma; and    -   h) binds to a different epitope than murine monoclonal antibody        64G12 (ECACC Deposit No. 92022605).

Preferred antibodies of the invention specifically bind to humaninterferon alpha receptor 1 and bind with a K_(D) of 1×10⁻⁸ M or greateraffinity, or 1×10⁻⁹ M or greater affinity, or 5×10⁻¹⁰ M or greateraffinity or 2×10⁻¹⁰ M or greater affinity

In one aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 4-34 or 5-51 gene, wherein the antibody specifically binds tohuman interferon alpha receptor 1. In another aspect, the inventionpertains to an isolated monoclonal antibody, or an antigen-bindingportion thereof, comprising a light chain variable region that is theproduct of or derived from a human V_(K) L18 or A27 gene, wherein theantibody specifically binds to human interferon alpha receptor 1. In yetanother aspect, the invention pertains to an isolated human monoclonalantibody, or antigen-binding portion thereof, comprising:

-   -   (a) a heavy chain variable region that is the product of or        derived from a human V_(H) 4-34 or 5-51 gene; and    -   (b) a light chain variable region that is the product of or        derived from a human Vk L18 or A27 gene;    -   wherein the antibody specifically binds to human interferon        alpha receptor 1.

In preferred embodiments, the antibody comprises a heavy chain variableregion of a human V_(H) 4-34 gene and a light chain variable region of ahuman V_(K) L18 gene or the antibody comprises a heavy chain variableregion of a human V_(H) 5-51 gene and a light chain variable region of ahuman V_(K) A27 gene.

In another aspect, the invention provides an isolated human monoclonalantibody, or antigen-binding portion thereof, comprising:

-   -   a human heavy chain variable region comprising CDR1, CDR2, and        CDR3 sequences; and a human light chain variable region        comprising CDR1, CDR2, and CDR3 sequences, wherein:    -   (a) the human heavy chain variable region CDR3 sequence        comprises an amino acid sequence selected from the group        consisting of amino acid sequences of SEQ ID NO: 9, 10, 11, and        12, and conservative modifications thereof;    -   (b) the human light chain variable region CDR3 sequence        comprises an amino acid sequence selected from the group        consisting of amino acid sequence of SEQ ID NO:21, 22, 23, and        24, and conservative modifications thereof;    -   (c) the antibody specifically binds human interferon alpha        receptor 1 with a binding affinity of at least 1×10⁻⁸ M or        greater affinity; and    -   (d) the antibody inhibits the biological activity of at least        one Type I interferon.

Preferably, the human heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NO: 5, 6, 7, and 8, and conservativemodifications thereof; and the human light chain variable region CDR2sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NO: 17, 18, 19, and 20, andconservative modifications thereof. Preferably, the human heavy chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NO: 1, 2, 3,and 4, and conservative modifications thereof; and the human light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NO: 13, 14,15, and 16, and conservative modifications thereof.

In another aspect, the invention pertains to an isolated humanmonoclonal antibody, or antigen-binding portion thereof, comprising ahuman heavy chain variable region and a human light chain variableregion, wherein:

-   -   (a) the human heavy chain variable region comprises an amino        acid sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NO: 25,        26, 27, and 28;    -   (b) the human light chain variable region comprises an amino        acid sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NO: 29,        30, 31, and 32;    -   (c) the antibody specifically binds human interferon alpha        receptor 1 with a binding affinity of at least 1×10⁻⁸ M or        greater affinity; and    -   (d) the antibody inhibits the biological activity of at least        one Type I interferon.

Preferred antibodies of the invention include isolated human monoclonalantibodies, or antigen-binding portions thereof, comprising:

-   -   (a) a human heavy chain variable region CDR1 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        1, 2, 3, and 4;    -   (b) a human heavy chain variable region CDR2 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        5, 6, 7, and 8;    -   (c) a human heavy chain variable region CDR3 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        9, 10, 11, and 12;    -   (d) a human light chain variable region CDR1 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        13, 14, 15, and 16;    -   (e) a human light chain variable region CDR2 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        17, 18, 19, and 20; and    -   (f) a human light chain variable region CDR3 comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        21, 22, 23, and 24;    -   wherein the antibody specifically binds human interferon alpha        receptor 1 with a binding affinity of at least 1×10⁻⁸ M or        greater affinity.

Preferred combinations of CDR regions include the following:

-   -   (a) a human heavy chain variable region CDR1 comprising SEQ ID        NO: 1;    -   (b) a human heavy chain variable region CDR2 comprising SEQ ID        NO:5;    -   (c) a human heavy chain variable region CDR3 comprising SEQ ID        NO:9;    -   (d) a human light chain variable region CDR1 comprising SEQ ID        NO:13;    -   (e) a human light chain variable region CDR2 comprising SEQ ID        NO:17; and    -   (f) a human light chain variable region CDR3 comprising SEQ ID        NO:21.    -   (a) a human heavy chain variable region CDR1 comprising SEQ ID        NO:2;    -   (b) a human heavy chain variable region CDR2 comprising SEQ ID        NO:6;    -   (c) a human heavy chain variable region CDR3 comprising SEQ ID        NO: 10;    -   (d) a human light chain variable region CDR1 comprising SEQ ID        NO:14;    -   (e) a human light chain variable region CDR2 comprising SEQ ID        NO:18; and    -   (f) a human light chain variable region CDR3 comprising SEQ ID        NO:22.    -   (a) a human heavy chain variable region CDR1 comprising SEQ ID        NO:3;    -   (b) a human heavy chain variable region CDR2 comprising SEQ ID        NO:7;    -   (c) a human heavy chain variable region CDR3 comprising SEQ ID        NO: 11;    -   (d) a human light chain variable region CDR1 comprising SEQ ID        NO:15;    -   (e) a human light chain variable region CDR2 comprising SEQ ID        NO:19; and    -   (f) a human light chain variable region CDR3 comprising SEQ ID        NO:23.    -   (a) a human heavy chain variable region CDR1 comprising SEQ ID        NO:4;    -   (b) a human heavy chain variable region CDR2 comprising SEQ ID        NO:8;    -   (c) a human heavy chain variable region CDR3 comprising SEQ ID        NO: 12;    -   (d) a human light chain variable region CDR1 comprising SEQ ID        NO: 16;    -   (e) a human light chain variable region CDR2 comprising SEQ ID        NO:20; and    -   (f) a human light chain variable region CDR3 comprising SEQ ID        NO:24.

Other preferred antibodies of the invention include isolated humanmonoclonal antibodies, or antigen binding portions thereof, comprising:

-   -   (a) a human heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 25,        26, 27, and 28; and    -   (b) a human light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 29,        30, 31, and 32;    -   wherein the antibody specifically binds human interferon alpha        receptor 1 with a binding affinity of at least 1×10⁻⁸ M or        greater affinity.

Preferred combinations of heavy and light chains include the following:

-   -   (a) a human heavy chain variable region comprising the amino        acid sequence of SEQ ID NO:25; and    -   (b) a human light chain variable region comprising the amino        acid sequence of SEQ ID NO:29.    -   (a) a human heavy chain variable region comprising the amino        acid sequence of SEQ ID NO:26; and    -   (b) a human light chain variable region comprising the amino        acid sequence of SEQ ID NO:30.    -   (a) a human heavy chain variable region comprising the amino        acid sequence of SEQ ID NO:27; and    -   (b) a human light chain variable region comprising the amino        acid sequence of SEQ ID NO:31.    -   (a) a human heavy chain variable region comprising the amino        acid sequence of SEQ ID NO:28; and    -   (b) a human light chain variable region comprising the amino        acid sequence of SEQ ID NO:32.

Another aspect of the invention pertains to antibodies that compete forbinding to IFNAR-1 with a reference antibody provided by the invention.Accordingly, in another embodiment, the invention provides:

-   -   an isolated monoclonal antibody, or antigen binding portion        thereof, wherein the antibody cross-competes for binding to        human interferon alpha receptor 1 with a reference antibody,        wherein the reference antibody is selected from the group        consisting of:    -   a) an antibody comprising a heavy chain variable region        comprising the amino acid sequence of SEQ ID NO: 25; and a light        chain variable region comprising the amino acid sequence of SEQ        ID NO: 29;    -   b) an antibody comprising a heavy chain variable region        comprising the amino acid sequence of SEQ ID NO: 26; and a light        chain variable region comprising the amino acid sequence of SEQ        ID NO: 30;    -   c) an antibody comprising a heavy chain variable region        comprising the amino acid sequence of SEQ ID NO: 27; and a light        chain variable region comprising the amino acid sequence of SEQ        ID NO: 31; and    -   d) an antibody comprising a heavy chain variable region        comprising the amino acid sequence of SEQ ID NO: 28; and a light        chain variable region comprising the amino acid sequence of SEQ        ID NO: 32.

In certain embodiments, the invention provides a human antibody, orantigen-binding portion thereof, wherein the antibody does not bind thesame epitope as (i.e., does not cross-compete with) mouse monoclonalantibody 64G12 (ECACC Deposit No. 92022605).

The antibodies of the invention can be of any isotype. Preferredantibodies are of the IgG1, IgG3 or IgG4 isotype. The antibodies of theinvention can be full-length antibodies comprising variable and constantregions, or they can be antigen-binding fragments thereof, such as asingle chain antibody, or a Fab or Fab′2 fragment.

The invention also provides an immunoconjugate comprising an antibody ofthe invention, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. Theinvention also provides a bispecific molecule comprising an antibody, orantigen-binding portion thereof, of the invention, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of the invention and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by theinvention, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Moreover, theinvention provides a transgenic mouse comprising human immunoglobulinheavy and light chain transgenes, wherein the mouse expresses anantibody of the invention, as well as hybridomas prepared from such amouse, wherein the hybridoma produces the antibody of the invention.

The invention also provides methods for making “second generation”anti-IFNAR-1 antibodies based on the sequences of the anti-IFNAR-1antibodies provided herein. For example, the invention provides a methodfor preparing an anti-IFNAR-1 antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence that is selected from the        group consisting of SEQ ID NOs: 1, 2, 3 and 4, a CDR2 sequence        that is selected from the group consisting of SEQ ID NOs: 5, 6,        7, and 8; and a CDR3 sequence that is selected from the group        consisting of SEQ ID NOs: 9, 10, 11, and 12; or (ii) a light        chain variable region antibody sequence comprising a CDR1        sequence that is selected from the group consisting of SEQ ID        NOs: 13, 14, 15, and 16, a CDR2 sequence that is selected from        the group consisting of SEQ ID NOs: 17, 18, 19, and 20 and a        CDR3 sequence that is selected from the group consisting of SEQ        ID NOs: 21, 22, 23, and 24;    -   (b) altering at least one amino acid residue within at least one        variable region antibody sequence, said sequence being selected        from the heavy chain variable region antibody sequence and the        light chain variable region antibody sequence, to create at        least one altered antibody sequence; and    -   (c) expressing the altered antibody sequence as a protein.

The invention also provides a method for inhibiting biological activityof a type I interferon on a cell expressing interferon alpha receptor 1comprising contacting the cell with the antibody of the invention, suchthat the biological activity of the type I interferon is inhibited. Theinvention also provides a method of treating a type Iinterferon-mediated disease or disorder in a subject in need oftreatment comprising administering to the subject the antibody, orantigen-binding portion thereof, of the invention, such that the type-Iinterferon mediated disease in the subject is treated. The type Iinterferon-mediated disease can be, for example, an interferonalpha-mediated disease.

Examples of disease or disorders that can be treated using the methodsof the invention include systemic lupus erythematosus, insulin dependentdiabetes mellitus, inflammatory bowel disease, multiple sclerosis,psoriasis, autoimmune thyroiditis, rheumatoid arthritis,glomerulonephritis, HIV infection, AIDS, transplant rejection and graftversus host disease.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all references, Genbank entries,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 33) and amino acidsequence (SEQ ID NO: 25) of the heavy chain variable region of the 3F11human monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 5)and CDR3 (SEQ ID NO: 9) regions are delineated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 37) and amino acidsequence (SEQ ID NO: 29) of the light chain variable region of the 3F11human monoclonal antibody. The CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO:17) and CDR3 (SEQ ID NO: 21) regions are delineated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 34) and amino acidsequence (SEQ ID NO: 26) of the heavy chain variable region of the 4G5human monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 6)and CDR3 (SEQ ID NO: 10) regions are delineated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 38) and amino acidsequence (SEQ ID NO: 30) of the light chain variable region of the 4G5human monoclonal antibody. The CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 22) regions are delineated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 35) and amino acidsequence (SEQ ID NO: 27) of the heavy chain variable region of the 11E2human monoclonal antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 7)and CDR3 (SEQ ID NO: 11) regions are delineated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 39) and amino acidsequence (SEQ ID NO: 31) of the light chain variable region of the 11E2human monoclonal antibody. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO: 23) regions are delineated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 36) and amino acidsequence (SEQ ID NO: 28) of the heavy chain variable region of the 9D4human monoclonal antibody. The CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 8)and CDR3 (SEQ ID NO: 12) regions are delineated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 40) and amino acidsequence (SEQ ID NO: 32) of the light chain variable region of the 9D4human monoclonal antibody. The CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO: 24) regions are delineated.

FIG. 5 shows the alignment of the amino acid sequence of the heavy chainvariable region of 3F11 with the human germline V_(H) 4-34 amino acidsequence (SEQ ID NO: 41).

FIG. 6 shows the alignment of the amino acid sequence of the heavy chainvariable region of 4G5 with the human germline V_(H) 4-34 amino acidsequence (SEQ ID NO: 41).

FIG. 7 shows the alignment of the amino acid sequence of the heavy chainvariable region of 11E2 and 9D4 with the human germline V_(H) 5-51 aminoacid sequence (SEQ ID NO: 42).

FIG. 8 shows the alignment of the amino acid sequence of the light chainvariable region of 3F11 with the human germline V_(k) L18 amino acidsequence (SEQ ID NO: 43).

FIG. 9 shows the alignment of the amino acid sequence of the light chainvariable region of 4G5 with the human germline V_(k) L18 amino acidsequence (SEQ ID NO: 43).

FIG. 10 shows the alignment of the amino acid sequence of the lightchain variable region of 11E2 and 9D4 with the human germline V_(k) A27amino acid sequence (SEQ ID NO: 44).

FIG. 11 is a graph showing the results of experiments demonstrating thatthe human monoclonal antibody, 3F11, directed against human IFNAR-1,does not compete with the mouse monoclonal antibody 64G12 for binding toIFNAR-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated monoclonal antibodies thatbind to Interferon alpha receptor 1 (IFNAR-1) and that are capable ofblocking the action of type I interferons. The invention providesisolated antibodies, methods of making such antibodies, immunoconjugatesand bispecific molecules comprising such antibodies and pharmaceuticalcompositions containing the antibodies, immunoconjugates or bispecificmolecules of the invention. The invention also relates to methods ofusing the antibodies to inhibit the binding of a type I interferon toIFNAR-1 on a cell expressing IFNAR-1, for example, in the treatment ofimmune mediated disorders, including autoimmune disorders, transplantrejection and Graft Versus Host Disease (GVHD), in a subject.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “Interferon alpha receptor-1,” “IFNAR-1,” and “IFNAR-1antigen” are used interchangeably, and include variants, isoforms,species homologs of human IFNAR-1, and analogs having at least onecommon epitope with IFNAR-1. Accordingly, human antibodies of theinvention may, in certain cases, cross-react with IFNAR-1 from speciesother than human, or other proteins which are structurally related tohuman IFNAR-1 (e.g., human IFNAR-1 homologs). In other cases, theantibodies may be completely specific for human IFNAR-1 and not exhibitspecies or other types of cross-reactivity.

The complete cDNA sequence of human IFNAR-1 has the Genbank accessionnumber NM_000629.

The term “type I interferon” as used herein is intended to refer tomembers of the type I interferon family of molecules that are ligandsfor IFNAR-1 (i.e., members of the type I interferon family of moleculesthat are capable of binding IFNAR-1). Examples of type I interferonligands are interferon alpha 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17,21, interferon beta and interferon omega.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present invention is the IFNAR-1 receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IFNAR-1). It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds IFNAR-1 is substantially free of antibodies that specifically bindantigens other than IFNAR-1). An isolated antibody that specificallybinds IFNAR-1 may, however, have cross-reactivity to other antigens,such as IFNAR-1 molecules from other species. Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10⁻⁷ M or less, and binds to the predeterminedantigen with a K_(D) that is at least two-fold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to an antibody having a KDof 10⁻⁷ M or less, more preferably 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-IFNAR-1 Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to IFNAR-1, preferably human IFNAR-1.Additionally, the antibodies may cross react with IFNAR-1 from one ormore non-human primates, such as cynomolgus monkey and/or rhesus monkey.Preferably, an antibody of the invention binds to IFNAR-1 with highaffinity, for example with a K_(D) of 10⁻⁷ M or less, more preferablywith a K_(D) of 10⁻⁸ M or less or 10⁻⁹ M or less or even 5×10⁻¹⁰ M orless or 2×10⁻¹⁰ M or less.

Furthermore, the antibodies of the invention are capable of inhibitingthe biological activity of type 1 interferons. The antibodies inhibitthe biological activity of at least one type I interferon, andpreferably inhibit the biological activity of multiple type Iinterferons (i.e., at least two, more preferably at least three, or atleast four, or at least five, or at least six, or at least seven, or atleast eight, or at least nine, or at least ten, or at least 11, or atleast 12, or at least 13 or at least 14 or at least 15, differentsubtypes of type I interferon). In a preferred embodiment, the antibodyinhibits the biological activity of the following type I interferons:α1, α 2a, α 2b, α 4, α 5, α 6, α 7, α 8, α 10, α 14, α 16, α 17, α 21,beta and omega. In other preferred embodiments, the antibody inhibitsthe activity of lymphoblastoid IFN and/or leukocyte IFN.

The ability of an antibody to inhibit the biological activity of type Iinterferons can be examined in one or more assays established in theart. Non-limiting examples include inhibition of Type I IFN-mediatedinhibition of Daudi cell proliferation, inhibition of Type I IFN-inducedexpression of IP-10 by peripheral blood mononuclear cells (PBMC),inhibition of dendritic cell development mediated by Systemic LupusErythematosus (SLE) plasma, and inhibition of the anti-viral activity ofType I IFN. At antibody “inhibits the biological activity of type Iinterferons” if it inhibits the activity by at least 20%, morepreferably by at least 30%, even more preferably by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90%, ascompared to a non-specific, control antibody.

In preferred embodiments, the antibody inhibits the activity of IFN α 2bin a Daudi cell proliferation assay, inhibits the activity of IFN omegain a Daudi cell proliferation assay, inhibits IP-10 secretion by PBMCinduced by IFN α 2b or IFN omega, and/or inhibits dendritic celldevelopment mediated by SLE plasma.

In another preferred embodiment, the antibody does not cross-competewith (i.e., binds to a different epitope than) the murine anti-IFNAR-1antibody 64G12 (deposited as ECACC Deposit No. 92022605).

Assays to evaluate the functional activities of anti-IFNAR antibodiesare described in further detail in the Examples. Preferred antibodies ofthe invention exhibit at least one, more preferably two, three, four,five or more, of the following properties:

-   -   a) specifically binds to IFNAR1 (preferably human IFNAR1);    -   b) binds to IFNAR1 with high affinity, such as a K_(D) of 1×10⁻⁸        M or greater affinity;    -   c) inhibits the biological activity of multiple Type I        interferons;    -   d) inhibits the activity of IFN α 2b in a Daudi cell        proliferation assay;    -   e) inhibits the activity of IFN omega in a Daudi cell        proliferation assay;    -   f) inhibits IP-10 secretion by peripheral blood mononuclear        cells induced by IFN α 2b;    -   g) inhibits IP-10 secretion by peripheral blood mononuclear        cells induced by IFN omega;    -   h) inhibits dendritic cell development mediated by Systemic        Lupus Erythematosus plasma; and    -   i) binds to a different epitope than (i.e., does not        cross-compete with) murine monoclonal antibody 64G12 (ECACC        Deposit No. 92022605).

Any combination of the above-described functional features, and/or thefunctional features as described in the Examples, may be exhibited by anantibody of the invention.

Monoclonal Antibody 3F11, 4G5, 11E2, and 9D4

Preferred antibodies of the invention are the human monoclonalantibodies 3F11, 4G5, 11E2, and 9D4, isolated and structurallycharacterized as described in the Examples. The V_(H) amino acidsequences of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 25, 26,27, and 28, respectively. The V_(L) amino acid sequences of 3F11, 4G5,11E2, and 9D4 are shown in SEQ ID NOs: 29, 30, 31, and 32, respectively.

Given that each of these antibodies can bind to IFNAR-1, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-IFNAR-1binding molecules of the invention. IFNAR-1 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedherein (e.g., ELISAs) and/or using the type I IFN functional inhibitionassays described in the Examples. Preferably, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence. For example, the V_(H) and V_(L) sequences of 3F11 and 4G5 areparticularly amenable for mixing and matching, since these antibodiesuse V_(H) and V_(L) sequences derived from the same germline sequences(V_(H) 4-34 and V_(k) L18) and thus they exhibit structural similarity.In addition, the V_(H) and V_(L) sequences of 11E2 and 9D4 areparticularly amenable for mixing and matching, since these antibodiesuse V_(H) and V_(L) sequences derived from the same germline sequences(V_(H) 5-51 and V_(k) A27) and thus they exhibit structural similarity.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 25,        26, 27, and 28; and    -   (b) a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 29,        30, 31, and 32;    -   wherein the antibody specifically binds IFNAR-1.

Preferred heavy and light chain combinations include:

-   -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 25; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 29; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 26; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 30; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 27; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 31; or    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 28; and (b) a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 3F11, 4G5, 11E2,and 9D4, or combinations thereof. The amino acid sequences of the V_(H)CDR1s of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 1, 2, 3, and4. The amino acid sequences of the V_(H) CDR2s of 3F11, 4G5, 11E2, and9D4 are shown in SEQ ID NOs: 5, 6, 7, and 8. The amino acid sequences ofthe V_(H) CDR3s of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 9,10, 11, and 12. The amino acid sequences of the V_(k) CDR1s of 3F11,4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 13, 14, 15, and 16. Theamino acid sequences of the V_(k) CDR2s of 3F11, 4G5, 11E2, and 9D4 areshown in SEQ ID NOs: 17, 18, 19, and 20. The amino acid sequences of theV_(k) CDR3s of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 21, 22,23, and 24. The CDR regions are delineated using the Kabat system(Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242).

Given that each of these antibodies can bind to IFNAR-1 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(k) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(k) CDR1, 2 and 3) to create otheranti-IFNAR-1 binding molecules of the invention. IFNAR-1 binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed above and in the Examples (e.g., ELISAs). Preferably, whenV_(H) CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3sequence from a particular V_(H) sequence is replaced with astructurally similar CDR sequence(s). Likewise, when V_(k) CDR sequencesare mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from aparticular V_(k) sequence preferably is replaced with a structurallysimilar CDR sequence(s). For example, the V_(H) CDR1s of 3F11 and 4G5share some structural similarity and therefore are amenable to mixingand matching. As another example, the V_(H) CDR1s of 11E2 and 9D4 sharesome structural similarity and therefore are amenable to mixing andmatching. As yet another example, the V_(K) CDR1s of 3F11 and 4G5 sharesome structural similarity. As yet another example, the V_(H) CDR1s of11E2 and 9D4 share some structural similarity. It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 3F11, 4G5, 11E2,and 9D4.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

-   -   (a) a heavy chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 1, 2,        3, and 4;    -   (b) a heavy chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 5, 6,        7, and 8;    -   (c) a heavy chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 9,        10, 11, and 12;    -   (d) a light chain variable region CDR1 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 13,        14, 15, and 16;    -   (e) a light chain variable region CDR2 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 17,        18, 19, and 20; and    -   (f) a light chain variable region CDR3 comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 21,        22, 23, and 24;    -   wherein the antibody specifically binds IFNAR-1.

In a preferred embodiment, the antibody comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 5;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 13;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 17;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 21.

In another preferred embodiment, the antibody comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 6;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 10;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 14;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 18;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 22.

In another preferred embodiment, the antibody comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 7;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 11;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 15;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 19;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 23.

In another preferred embodiment, the antibody comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 4;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 8;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 16;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 20;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 24.        Antibodies that Bind to the Same Epitope as 3F11, 4G5, 11E2, and        9D4

In another embodiment, the invention provides antibodies that bind tothe same epitope on human IFNAR-1 as the monoclonal antibodies 3F11,4G5, 11E2, or 9D4 (having V_(H) sequences as shown in SEQ ID NOs: 25,26, 27, and 28, respectively, and V_(L) sequences as shown in SEQ IDNOs: 29, 30, 31, 32, respectively). Such antibodies can be identifiedbased on their ability to cross-compete with 3F11, 4G5, 11E2, or 9D4 instandard IFNAR-1 binding assays. The ability of a test antibody toinhibit the binding of 3F11, 4G5, 11E2, or 9D4 to human IFNAR-1demonstrates that the test antibody can compete with 3F11, 4G5, 11E2, or9D4 for binding to human IFNAR-1 and thus binds to the same epitope onhuman IFNAR-1 as 3F11, 4G5, 11E2, or 9D4. In a preferred embodiment, theantibody that binds to the same epitope on human IFNAR-1 as 3F11, 4G5,11E2, or 9D4 is a human monoclonal antibody. Such human monoclonalantibodies can be prepared and isolated as described in the Examples.

In another preferred embodiment, the antibody binds to a differentepitope than (i.e., does not cross-compete with) the mouse monoclonalantibody 64G12 (ECACC Deposit No. 92022605).

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides anisolated anti-IFNAR-1 monoclonal antibody, or an antigen-binding portionthereof, wherein the antibody:

-   -   (a) comprises a heavy chain variable region of a human VH 4-34        or 5-51 gene;    -   (b) comprises a light chain variable region of a human Vk L18 or        A27 gene; and    -   (c) the antibody specifically binds to IFNAR-1.

Examples of antibodies having V_(H) and V_(K) of VH 4-34 and Vk L18,respectively, include 3F11 and 4G5. Examples of antibodies having V_(H)and V_(K) of VH 5-51 and Vk A27, respectively, include 11E2 and 9D4.

As used herein, a human antibody comprises heavy or light chain variableregions “of” or “derived from” or “the product of” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “of” or “derived from” or “the product of” a humangermline immunoglobulin sequence can be identified as such by comparingthe amino acid sequence of the human antibody to the amino acidsequences of human germline immunoglobulins and selecting the humangermline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the human antibody. A humanantibody that is “of” or “derived from” or “the product of” a particularhuman germline immunoglobulin sequence may contain amino aciddifferences as compared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodymay be at least 95%, or even at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-IFNAR-1 antibodies of the invention.For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

-   -   (a) the heavy chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 25,        26, 27, and 28;    -   (b) the light chain variable region comprises an amino acid        sequence that is at least 80% homologous to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 29,        30, 31, and 32;    -   (c) the antibody specifically binds to IFNAR-1 and exhibits at        least one of the functional properties described herein,        preferably several of the functional properties described        herein.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, or 40, followed bytesting of the encoded altered antibody for retained function (i.e., thefunctions set forth in (c), (d) and (e) above) using the functionalassays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,3F11, 4G5, 11E2, and 9D4), or conservative modifications thereof, andwherein the antibodies retain the desired functional properties of theanti-IFNAR-1 antibodies of the invention. Accordingly, the inventionprovides an isolated monoclonal antibody, or antigen binding portionthereof, comprising a heavy chain variable region comprising CDR1, CDR2,and CDR3 sequences and a light chain variable region comprising CDR1,CDR2, and CDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 9, 10, 11, and 12, and        conservative modifications thereof;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 21, 22, 23, and 24, and        conservative modifications thereof; and    -   (c) the antibody specifically binds to IFNAR-1 and exhibits at        least one of the functional properties described herein, more        preferably several of the functional properties described        herein.

In a further embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 5, 6, 7, and 8, and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 17, 18, 19, and 20, and conservativemodifications thereof. In a still further embodiment, the heavy chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 1, 2,3, and 4, and conservative modifications thereof; and the light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 13, 14,15, and 16, and conservative modifications thereof.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c), (d) and (e) above) usingthe functional assays described herein.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising: aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 3, and 4, SEQ ID NOs: 5, 6, 7, and 8 and SEQ ID NOs:9, 10, 11, and 12, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 sequences comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 13, 14, 15,and 16, SEQ ID NOs: 17, 18, 19, and 20 and SEQ ID NOs: 21, 22, 23, and24, respectively. Thus, such antibodies contain the V_(H) and V_(L) CDRsequences of monoclonal antibodies 3F11, 4G5, 11E2, or 9D4 yet maycontain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H) 4-34and V_(L) L18 framework sequences used by the 3F11 and 4G5 monoclonalantibodies, or the V_(H) 5-51 and V_(L) A27 framework sequences used bythe 11E2 and 9D4 monoclonal antibodies. The V_(H) CDR1, 2 and 3sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, andthe V_(L) CDR1, 2 and 3 sequences of SEQ ID NOs: 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, and 24 can be grafted onto framework regions thathave the same sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than five residues are altered within a CDR region arealtered.

Accordingly, in another embodiment, the invention provides isolatedanti-IFNAR-1 monoclonal antibodies, or antigen binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, 2, 3, and 4, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, 2, 3, and 4; (b) a V_(H) CDR2 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 5, 6, 7, and 8, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to an amino acid sequence selected from the group consisting ofSEQ ID NO: 5, 6, 7, and 8; (c) a V_(H) CDR3 region comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, 10,11, and 12, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to anamino acid sequence selected from the group consisting of SEQ ID NO: 9,10, 11, and 12; (d) a V_(L) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 13, 14, 15,and 16, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to an aminoacid sequence selected from the group consisting of SEQ ID NO: 13, 14,15, and 16; (e) a V_(L) CDR2 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 17, 18, 19, and 20, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to an amino acidsequence selected from the group consisting of SEQ ID NO: 17, 18, 19,and 20; and (f) a V_(L) CDR3 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 21, 22, 23, and 24, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to an amino acidsequence selected from the group consisting of SEQ ID NO: 17, 18, 19,and 20.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. For example, for 3F11, amino acidresidue #43 (within FR2) of V_(H) is a threonine whereas this residue inthe corresponding V_(H) 4-34 germline sequence is an alanine (see FIG.5). To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis orPCR-mediated mutagenesis (e.g., residue 43 of the V_(H) of 3F11 can be“backmutated” from threonine to alanine). As another example, for 4G5,amino acid residue #81 (within FR3) of V_(H) is an asparagine whereasthis residue in the corresponding V_(H) 4-34 germline sequence is alysine (see FIG. 6). To return the framework region sequences to theirgermline configuration, the somatic mutations can be “backmutated” tofrom asparagine to lysine. As another example, for 11E2 and 9D4, aminoacid residue #28 (within FR1) of V_(H) is an isoleucine whereas thisresidue in the corresponding V_(H) 5-51 germline sequence is a serine(see FIG. 7). To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to fromisoleucine to serine. Such “backmutated” antibodies are also intended tobe encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter it's glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hanai et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

Thus, in another aspect of the invention, the structural features ofanti-IFNAR-1 antibodies of the invention, e.g. 3F11, 4G5, 11E2, and 9D4are used to create structurally related anti-IFNAR-1 antibodies thatretain at least one functional property of the antibodies of theinvention, such as binding to IFNAR-1. For example, one or more CDRregions of 3F11, 4G5, 11E2, or 9D4, or mutations thereof, can becombined recombinantly with known framework regions and/or other CDRs tocreate additional, recombinantly-engineered, anti-IFNAR-1 antibodies ofthe invention, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the V_(H) and/or V_(L) sequencesprovided herein, or one or more CDR regions thereof. To create theengineered antibody, it is not necessary to actually prepare (i.e.,express as a protein) an antibody having one or more of the V_(H) and/orV_(L) sequences provided herein, or one or more CDR regions thereof.Rather, the information contained in the sequence(s) is used as thestarting material to create a “second generation” sequence(s) derivedfrom the original sequence(s) and then the “second generation”sequence(s) is prepared and expressed as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IFNAR-1 antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence selected from the group        consisting of SEQ ID NOs: 1, 2, 3 and 4, a CDR2 sequence        selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8        and/or a CDR3 sequence selected from the group consisting of SEQ        ID NOs: 9, 10, 11, and 12; and (ii) a light chain variable        region antibody sequence comprising a CDR1 sequence selected        from the group consisting of SEQ ID NOs: 13, 14, 15, and 16, a        CDR2 sequence selected from the group consisting of SEQ ID NOs:        17, 18, 19, and 20 and/or a CDR3 sequence selected from the        group consisting of SEQ ID NOs: 21, 22, 23, and 24;    -   (b) altering at least one amino acid residue within the first        antibody sequence and/or the second antibody sequence to create        at least one altered antibody sequence; and    -   (c) preparing the altered antibody sequence; and    -   (d) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-IFNAR-1 antibodies described herein, which functional propertiesinclude, but are not limited to:

-   -   (i) binding to IFNAR-1;    -   (ii) inhibiting the binding of type I interferons to IFNAR-1;    -   (iii) binding to live cells expressing human IFNAR-1;    -   (iv) binding to human IFNAR-1 with a K_(D) of 10⁻⁸ M or less        (e.g., 10⁻⁹ M or 10⁻¹⁰ M or less);    -   (v) binding to a unique epitope on IFNAR-1 (to eliminate the        possibility that monoclonal antibodies with complimentary        activities when used in combination would compete for binding to        the same epitope).

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein. Forexample, the ability of the antibody to bind IFNAR-1 can be determinedusing standard binding assays, such as those set forth in the Examples(e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-IFNAR-1 antibody coding sequence (e.g., 3F11, 4G5,11E2, or 9D4 coding sequence) and the resulting modified anti-IFNAR-1antibodies can be screened for binding activity and/or other functionalproperties as described herein. Mutational methods have been describedin the art. For example, PCT Publication WO 02/092780 by Short describesmethods for creating and screening antibody mutations using saturationmutagenesis, synthetic ligation assembly, or a combination thereof.Alternatively, PCT Publication WO 03/074679 by Lazar et al. describesmethods of using computational screening methods to optimizephysiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules of the invention are those encodingthe VH and VL sequences of the 3F11, 4G5, 11E2, and 9D4 monoclonalantibodies. DNA sequences encoding the 3F11 VH and VL sequences areshown in SEQ ID NOs: 33 and 37, respectively. DNA sequences encoding the4G5 VH and VL sequences are shown in SEQ ID NOs: 34 and 38,respectively. DNA sequences encoding the 11E2 VH and VL sequences areshown in SEQ ID NOs: 35 and 39, respectively. DNA sequences encoding the9D4 VH and VL sequences are shown in SEQ ID NOs: 36 and 40,respectively.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., el al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstIFNAR-1 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IFNAR-1 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IFNAR-1 antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-IFNAR-1antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or enriched preparation ofIFNAR-1 antigen and/or cells expressing IFNAR-1, as described byLonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al.(1996) Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884and WO 01/14424. Preferably, the mice will be 6-16 weeks of age upon thefirst infusion. For example, a purified or enriched preparation (5-50pig) of IFNAR-1 antigen can be used to immunize the human Ig miceintraperitoneally. In the event that immunizations using a purified orenriched preparation of IFNAR-1 antigen do not result in antibodies,mice can also be immunized with cells expressing IFNAR-1, e.g., a humanT-cell line, to promote immune responses.

Detailed procedures to generate fully human monoclonal antibodies toIFNAR-1 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-IFNAR-1 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12).

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×10⁵in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to IFNAR-1 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified IFNAR-1 at 0.25 μg/ml in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom IFNAR-1-immunized mice) are added to each well and incubated for1-2 hours at 37° C. The plates are washed with PBS/Tween and thenincubated with secondary reagent (e.g., for human antibodies, agoat-anti-human IgG Fc-specific polyclonal reagent) conjugated toalkaline phosphatase for 1 hour at 37° C. After washing, the plates aredeveloped with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650.Preferably, mice which develop the highest titers will be used forfusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with IFNAR-1 immunogen.Hybridomas that bind with high avidity to IFNAR-1 are subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-IFNAR-1 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-IFNAR-1 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using IFNAR-1 coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To demonstrate binding of monoclonal antibodies to live cells expressingIFNAR-1, flow cytometry can be used. Briefly, cell lines expressingIFNAR-1 (grown under standard growth conditions) are mixed with variousconcentrations of monoclonal antibodies in PBS containing 0.1% BSA and10% fetal calf serum, and incubated at 37° C. for 1 hour. After washing,the cells are reacted with Fluorescein-labeled anti-human IgG antibodyunder the same conditions as the primary antibody staining. The samplescan be analyzed by FACScan instrument using light and side scatterproperties to gate on single cells. An alternative assay usingfluorescence microscopy may be used (in addition to or instead of) theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence microscopy. This method allowsvisualization of individual cells, but may have diminished sensitivitydepending on the density of the antigen.

Anti-IFNAR-1 human IgGs can be further tested for reactivity withIFNAR-1 antigen by Western blotting. Briefly, cell extracts from cellsexpressing IFNAR-1 can be prepared and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens are transferred to nitrocellulose membranes, blockedwith 10% fetal calf serum, and probed with the monoclonal antibodies tobe tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugates

In another aspect, the present invention features an anti-IFNAR-1antibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunconjugates areestablished in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-IFNAR-1 antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for IFNAR-1 and asecond binding specificity for a second target epitope. In a particularembodiment of the invention, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific molecules capable ofbinding both to FcγR, FcαR or FcεR expressing effector cells (e.g.,monocytes, macrophages or polymorphonuclear cells (PMNs)), and to targetcells expressing IFNAR-1. These bispecific molecules target IFNAR-1expressing cells to effector cell and trigger Fc receptor-mediatedeffector cell activities, such as phagocytosis of an IFNAR-1 expressingcells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokinerelease, or generation of superoxide anion.

In an embodiment of the invention in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-IFNAR-1 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen.

Alternatively, the anti-enhancement factor portion can bind to an entitythat is different from the entity to which the first and second bindingspecificities bind. For example, the anti-enhancement factor portion canbind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1or other immune cell that results in an increased immune responseagainst the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγRI(CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFcγ binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of the invention because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-IFNAR-1 binding specificities, using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-IFNAR-1 antibody of the presentinvention combined with at least one other immunosuppressing agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-IFNAR-1antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 jμg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-IFNAR-1 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. In the case of, for example, Systemic Lupus Erythematosus(SLE), a therapeutically effective dose preferably prevents furtherdeterioration of physical symptoms associated with SLE, such as, forexample, pain, fatigue or weakness. A therapeutically effective dosepreferably also prevents or delays onset of SLE, such as may be desiredwhen early or preliminary signs of the disease are present. Likewise itincludes delaying chronic progression associated with SLE. Laboratorytests utilized in the diagnosis of SLE include chemistries, hematology,serology and radiology. Accordingly, any clinical or biochemical assaythat monitors any of the foregoing may be used to determine whether aparticular treatment is a therapeutically effective dose for treatingSLE. One of ordinary skill in the art would be able to determine suchamounts based on such factors as the subject's size, the severity of thesubject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994). Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies (and immunoconjugates and bispecific molecules) of thepresent invention have in vitro and in vivo diagnostic and therapeuticutilities. For example, these molecules can be administered to cells inculture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, totreat, prevent or diagnose a variety of disorders. The term “subject” asused herein in intended to includes human and non-human animals.Non-human animals includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles. The methods are particularlysuitable for treating human patients having a disorder associated withaberrant or inappropriate Type I interferon expression (e.g.,overexpression).

When antibodies to IFNAR-1 are administered together with another agent,the two can be administered in either order or simultaneously. Forexample, an anti-IFNAR-1 antibody of the invention can be used incombination with one or more of the following agents: anti-IFNαantibody, anti-IFNγ receptor antibody, soluble IFNγ receptor, anti-TNFantibody, anti-TNF receptor antibody and/or soluble TNF receptor (seee.g., U.S. Pat. No. 5,888,511). Furthermore, an anti-IFNAR-1 antibody ofinvention can be used in combination with a Flt3 ligand antagonist (seee.g., U.S. Application No. 2002/0160974).

In one embodiment, the antibodies (and immunoconjugates and bispecificmolecules) of the invention can be used to detect levels of IFNAR-1, orlevels of cells that express IFNAR-1. This can be achieved, for example,by contacting a sample (such as an in vitro sample) and a control samplewith the anti-IFNAR-1 antibody under conditions that allow for theformation of a complex between the antibody and IFNAR-1. Any complexesformed between the antibody and IFNAR-1 are detected and compared in thesample and the control. For example, standard detection methods,well-known in the art, such as ELISA and flow cytometic assays, can beperformed using the compositions of the invention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of IFNAR-1 (e.g., human IFNAR-1 antigen) in asample, or measuring the amount of IFNAR-1, comprising contacting thesample, and a control sample, with an antibody of the invention, or anantigen binding portion thereof, which specifically binds to IFNAR-1,under conditions that allow for formation of a complex between theantibody or portion thereof and IFNAR-1. The formation of a complex isthen detected, wherein a difference in complex formation between thesample compared to the control sample is indicative of the presence ofIFNAR-1 in the sample.

Also within the scope of the invention are kits comprising thecompositions (e.g., antibodies, human antibodies, immunoconjugates andbispecific molecules) of the invention and instructions for use. The kitcan further contain a least one additional reagent, or one or moreadditional antibodies of the invention (e.g., an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

IFNAR-1 is part of the cellular receptor for Type I interferons, andType I interferons are known to be immunoregulatory cytokines that areinvolved in, inter alia, T cell differentiation, antibody production andactivity and survival of memory T cells. Moreover, increased expressionof Type I interferons has been described in numerous autoimmunediseases, in HIV infection, in transplant rejection and in graft versushost disease (GVHD). Accordingly, the anti-IFNAR-1 antibodies (andimmunoconjugates and bispecific molecules) of the invention, whichinhibit the functional activity of Type I interferons, can be used in avariety of clinical indications involving aberrant or undesired Type Iinterferon activity. The invention, therefore, provides a method ofinhibiting a Type I interferon-mediated disease or disorder, wherein themethod comprises administering an antibody, or antigen-binding portionthereof, of the invention (or immunoconjugate or bispecific molecule ofthe invention) such that the Type I interferon-mediated disease ordisorder is treated.

Specific examples of autoimmune conditions in which the antibodies ofthe invention can be used include, but are not limited to, thefollowing: systemic lupus erythematosus (SLE), insulin dependentdiabetes mellitus (IDDM), inflammatory bowel disease (IBD) (includingCrohn's Disease, Ulcerative Colitis and Celiac's Disease), multiplesclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis(RA) and glomerulonephritis. Furthermore, the antibody compositions ofthe invention can be used for inhibiting or preventing transplantrejection or in the treatment of graft versus host disease (GVHD) or inthe treatment of HIV infection/AIDS.

High levels of IFNα have been observed in the serum of patients withsystemic lupus erythematosus (SLE) (see e.g., Kim et al. (1987) Clin.Exp. Immunol. 70:562-569). Moreover, administration of IFNα, for examplein the treatment of cancer or viral infections, has been shown to induceSLE (Garcia-Porrua et al. (1998) Clin. Exp. Rheumatol. 16:107-108).Accordingly, in another embodiment, the anti-IFNAR-1 antibodies of theinvention can be used in the treatment of SLE by administering theantibody to a subject in need of treatment. The antibody can be usedalone or in combination with other anti-SLE agents, such asnon-steroidal anti-inflammatory drugs (NSAIDs), analgesics,corticosteroids (e.g., predinisone, hydrocortisone), immunosuppressants(such as cyclophosphamide, azathioprine, and methotrexate),antimalarials (such as hydroxychloroquine) and biologic drugs thatinhibit the production of dsDNA antibodies (e.g., LJP 394).

IFN also has been implicated in the pathology of Type I diabetes. Forexample, the presence of immunoreactive IFNα in pancreatic beta cells ofType I diabetes patients has been reported (Foulis et al. (1987) Lancet2:1423-1427). Prolonged use of IFNα in anti-viral therapy also has beenshown to induce Type I diabetes (Waguri et al. (1994) Diabetes Res.Clin. Pract. 23:33-36). Accordingly, in another embodiment, theanti-IFNAR-1 antibodies of the invention can be used in the treatment ofType I diabetes by administering the antibody to a subject in need oftreatment. The antibody can be used alone or in combination with otheranti-diabetic agents, such as insulin.

Antibodies to IFNAR have been shown to be effective in an animal modelof inflammatory bowel disease (see U.S. Patent Application 60/465,155).Thus, the anti-IFNAR-1 antibodies of the invention can be used in thetreatment of inflammatory bowel disease (IBD), including ulcerativecolitis and Crohn's disease, by administering the antibody to a subjectin need of treatment. The antibody can be used alone or in combinationwith other anti-IBD agents, such as drugs containing mesalamine(including sulfasalazine and other agents containing 5-aminosalicylicacid (5-ASA), such as olsalazine and balsalazide), non-steroidalanti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g.,predinisone, hydrocortisone), TNF-inhibitors (including adilimumab(Humira®), etanercept (Enbrel®) and infliximab (Remicade®)),immunosuppressants (such as 6-mercaptopurine, azathioprine andcyclosporine A), and antibiotics.

Treatment with IFNα has also been observed to induce autoimmunethyroiditis (Monzani et al. (2004) Clin. Exp. Med. 3:199-210; Prummeland Laurberg (2003) Thyroid 13:547-551). Accordingly, in anotherembodiment, the anti-IFNAR antibodies of the invention can be used inthe treatment of autoimmune thyroid disease, including autoimmuneprimary hypothyroidism, Graves' Disease, Hashimoto's thyroiditis anddestructive thyroiditis with hypothyroidism, by administering theantibody to a subject in need of treatment. The antibody can be usedalone or in combination with other agents or treatments, such asanti-thyroid drugs, radioactive iodine and subtotal thyroidectomy.

Increased levels of type I interferons, especially IFB-β, have beenobserved in the serum of patients with RA (see e.g., Hertzog et al.(1988) Clin. Immunol. Immunopath. 48:192). Thus, in an embodiment, theanti-IFNAR-1 antibodies of the present invention can be used in thetreatment of RA by administering the antibody to a subject in need ofsuch treatment. The antibody can be used alone or in combination withone or more other anti-RA agent, such as a non-steroidalanti-inflammatory drug (NSAID), a COX-2 inhibitor, an analgesic, acorticosteroid (e.g., predinisone, hydrocortisone), gold, animmunosuppressant (e.g., methotrexate), a B-cell depletion agent (e.g.,Rituxan™), a B-cell agonist (e.g., LymphoStat-B™) and an anti-TNF-αagent (e.g., EMBREL™, HUMIRA® and REMICADE™).

Administration of IFNα has been reported to exacerbate psoriasis.Accordingly, in another embodiment, the anti-IFNAR-1 antibodies of theinvention can be used in the treatment of psoriasis and psoriaticarthritis by administering the antibody to a subject in need of suchtreatment. The antibody can be used alone or in combination with one ormore other anti-psoriasis treatments such as phototherapy, topicaltherapy (e.g., topical glucocorticoids), or systemic therapy (e.g.,methotrexate, a synthetic retinoid, cyclosporine), an anti-TNF-α agent(e.g., EMBREL™, HUMIRA® and REMICADE™), and a T-cell inhibitor (e.g.,Raptiva™).

High levels of IFNa also have been observed in the circulation ofpatients with HIV infection and its presence is a predictive marker ofAIDS progression (DeStefano et al. (1982) J. Infec. Disease 146:451;Vadhan-Raj et al. (1986) Cancer Res. 46:417). Thus, in anotherembodiment, an anti-IFNAR-1 antibody of the invention is used in thetreatment of HIV infection or AIDS by administering the antibody to asubject in need of treatment. The antibody can be used alone or incombination with other anti-HIV agents, such as nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, protease inhibitors and fusion inhibitors.

Antibodies to IFNAR-1 have been demonstrated to be effective ininhibiting allograft rejection and prolonging allograft survival (seee.g., Tovey et al. (1996) J. Leukoc. Biol. 59:512-517; Benizri et al.(1998) J. Interferon Cytokine Res. 18:273-284). Accordingly, theanti-IFNAR-1 antibodies of the invention also can be used in transplantrecipients to inhibit allograft rejection and/or prolong allograftsurvival. The invention provides a method of inhibiting transplantrejection by administering an anti-IFNAR-1 antibody of the invention toa transplant recipient in need of treatment. Examples of tissuetransplants that can be treated include, but are not limited to, liver,lung, kidney, heart, small bowel, and pancreatic islet cells, as well asthe treatment of graft versus host disease (GVHD). The antibody can beused alone or in combination with other agents for inhibiting transplantrejection, such as immunosuppressive agents (e.g., cyclosporine,azathioprine, methylprednisolone, prednisolone, prednisone,mycophenolate mofetil, sirilimus, rapamycin, tacrolimus), anti-infectiveagents (e.g., acyclovir, clotrimazole, ganciclovir, nystatin,trimethoprimsulfarnethoxazole), diuretics (e.g., bumetanide, furosemide,metolazone) and ulcer medications (e.g., cimetidine, famotidine,lansoprazole, omeprazole, ranitidine, sucralfate).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

Example 1: Generation of Human Monoclonal Antibodies Against IFNAR-1

Antigen

Soluble IFNAR-1, containing the extracellular domain of IFNAR-1 wasgenerated by recombinant methods and used as antigen for immunization.

Transgenic HuMab Mice

Fully human monoclonal antibodies to IFNAR-1 were prepared using HCo7,HCo12, and HCo7×HCo12 strains of HuMab transgenic mice, each of whichexpress human antibody genes. In each of these mouse strains, theendogenous mouse kappa light chain gene has been homozygously disruptedas described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenousmouse heavy chain gene has been homozygously disrupted as described inExample 1 of PCT Publication WO 01/09187. Each of these mouse strainscarries a human kappa light chain transgene, KCo5, as described inFishwild et al. (1996) Nature Biotechnology 14:845-851. The HCo7 straincarries the HCo7 human heavy chain transgene as described in U.S. Pat.Nos. 5,545,806; 5,625,825; and 5,545,807. The HCo12 strain carries theHCo12 human heavy chain transgene as described in Example 2 of PCTPublication WO 01/09187. The HCo7×HCo12 stain carries both the HCo7 andthe HCo12 transgenes and was made by breeding the two strains together.

HuMab Mice Immunizations:

To generate fully human monoclonal antibodies to IFNAR-1, HuMab micewere immunized with purified recombinant IFNAR-1 as antigen. Generalimmunization schemes for HuMab mice are described in Lonberg, N. et al(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851 and PCT Publication WO 98/24884. The mice were6-16 weeks of age upon the first infusion of antigen. A purifiedrecombinant preparation (5-50 μg) of soluble IFNAR-1 antigen was used toimmunize the HuMab mice intraperitonealy, subcutaneously (Sc) or viafootpad injection.

Transgenic mice were immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant either intraperitonealy (IP), subcutaneously(Sc) or via footpad (FP), followed by 3-21 days IP, Sc or FPimmunization (up to a total of 11 immunizations) with the antigen inincomplete Freund's or Ribi adjuvant. The immune response was monitoredby retroorbital bleeds. The plasma was screened by ELISA (as describedbelow), and mice with sufficient titers of anti-IFNAR-1 humanimmunoglobulin were used for fusions. Mice were boosted intravenouslywith antigen 3 and 2 days before sacrifice and removal of the spleen.Typically, 10-35 fusions for each antigen were performed. Several dozenmice were immunized for each antigen.

Selection of HuMab Mice Producing Anti-IFNAR-1 Antibodies:

To select HuMab mice producing antibodies that bound IFNAR-1, sera fromimmunized mice was tested by ELISA as described by Fishwild, D. et al.(1996). Briefly, microtiter plates were coated with purified recombinantIFNAR-1 from E. coli at 1-2 μg/ml in PBS, 50 μl/wells incubated 4° C.overnight then blocked with 200 μl/well of 5% chicken serum in PBS/Tween(0.05%). Dilutions of plasma from IFNAR-1-immunized mice were added toeach well and incubated for 1-2 hours at ambient temperature. The plateswere washed with PBS/Tween and then incubated with a goat-anti-human IgGFc polyclonal antibody conjugated with horseradish peroxidase (HRP) for1 hour at room temperature. After washing, the plates were developedwith ABTS substrate (Sigma, A-1888, 0.22 mg/ml) and analyzed byspectrophotometer at OD 415-495. Mice that developed the highest titersof anti-IFNAR-1 antibodies were used for fusions. Fusions were performedas described below and hybridoma supernatants were tested foranti-IFNAR-1 activity by ELISA.

Generation of Hybridomas Producing Human Monoclonal Antibodies toIFNAR-1:

The mouse splenocytes, isolated from the HuMab mice, were fused with PEGto a mouse myeloma cell line based upon standard protocols. Theresulting hybridomas were then screened for the production ofantigen-specific antibodies. Single cell suspensions of spleniclymphocytes from immunized mice were fused to one-fourth the number ofSP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG(Sigma). Cells were plated at approximately 1×10⁵/well in flat bottommicrotiter plate, followed by about two week incubation in selectivemedium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63)conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013,with high glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES,0.055 mM 2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, CRLP-7185). After 1-2 weeks, cells were cultured in medium in which the HATwas replaced with HT. Individual wells were then screened by ELISA(described above) for human anti-IFNAR-1 monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. The antibody secreting hybridomas were replated, screenedagain and, if still positive for human IgG, anti-IFNAR-1 monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Hybridoma clones 3F11, 4G5, 11E2, and 9D4 were selected for furtheranalysis.

Example 2: Structural Characterization of Human Monoclonal Antibodies3F11, 4G5, 11E2, and 9D4

The cDNA sequences encoding the heavy and light chain variable regionsof the 3F11, 4G5, 11E2, and 9D4 monoclonal antibodies were obtained fromthe 3F11, 4G5, 11E2, and 9D4 hybridomas, respectively, using standardPCR techniques and were sequenced using standard DNA sequencingtechniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 3F11 are shown in FIG. 1A and in SEQ ID NO: 33 and 25,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 3F11 are shown in FIG. 1B and in SEQ ID NO: 37 and 29,respectively.

Comparison of the 3F11 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 3F11 heavy chain utilizes a VH segment from human germline VH 4-34,an undetermined D segment, and a JH segment from human germline JH 6b.The alignment of the 3F11 VH sequence to the germline VH 4-34 sequenceis shown in FIG. 5. Further analysis of the 3F11 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 1A and 5, andin SEQ ID NOs: 1, 5 and 9, respectively.

Comparison of the 3F11 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 3F11 light chain utilizes a VL segment from human germline VK L18and a JK segment from human germline JK 5. The alignment of the 3F11 VLsequence to the germline VK L18 sequence is shown in FIG. 8. Furtheranalysis of the 3F11 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 1B and 8, and in SEQ ID NOs:13, 17 and 21,respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 4G5 are shown in FIG. 2A and in SEQ ID NO: 34 and 26,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 4G5 are shown in FIG. 2B and in SEQ ID NO: 38 and 30,respectively.

Comparison of the 4G5 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 4G5 heavy chain utilizes a VH segment from human germline VH 4-34,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 4G5 VH sequence to the germline VH 4-34 sequence isshown in FIG. 6. Further analysis of the 4G5 VH sequence using the Kabatsystem of CDR region determination led to the delineation of the heavychain CDR1, CDR2 and CD3 regions as shown in FIGS. 2A and 6, and in SEQID NOs: 2, 6 and 10, respectively.

Comparison of the 4G5 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 4G5 light chain utilizes a VL segment from human germline VK L18 anda JK segment from human germline JK 2. The alignment of the 4G5 VLsequence to the germline VK L18 sequence is shown in FIG. 9. Furtheranalysis of the 4G5 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 2B and 9, and in SEQ ID NOs:14, 18 and 22,respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 11E2 are shown in FIG. 3A and in SEQ ID NO: 35 and 27,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 11E2 are shown in FIG. 3B and in SEQ ID NO: 39 and 31,respectively.

Comparison of the 11E2 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 11E2 heavy chain was derived from, or is highly similar to, a VHsegment from human germline VH 5-51, an undetermined D segment, and a JHsegment from human germline JH 4b. The alignment of the 11E2 VH sequenceto the germline VH 5-51 sequence is shown in FIG. 7. Further analysis ofthe 11E2 VH sequence using the Kabat system of CDR region determinationled to the delineation of the heavy chain CDR1, CDR2 and CD3 regions asshown in FIGS. 3A and 7, and in SEQ ID NOs: 3, 7 and 11, respectively.

Comparison of the 11E2 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 11E2 light chain utilizes a VL segment from human germline VK A27and a JK segment from human germline JK 5. The alignment of the 11E2 VLsequence to the germline VK A27 sequence is shown in FIG. 10. Furtheranalysis of the 11E2 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 10, and in SEQ ID NOs:15, 19 and23, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 9D4 are shown in FIG. 4A and in SEQ ID NO: 36 and 28,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 9D4 are shown in FIG. 4B and in SEQ ID NO: 40 and 32,respectively.

Comparison of the 9D4 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 9D4 heavy chain was derived from, or is highly similar to, a VHsegment from human germline VH 5-51, an undetermined D segment, and a JHsegment from human germline JH 4b. The alignment of the 9D4 VH sequenceto the germline VH 5-51 sequence is shown in FIG. 7. Further analysis ofthe 9D4 VH sequence using the Kabat system of CDR region determinationled to the delineation of the heavy chain CDR1, CDR2 and CD3 regions asshown in FIGS. 4A and 7, and in SEQ ID NOs: 4, 8 and 12, respectively.

Comparison of the 9D4 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 9D4 light chain utilizes a VL segment from human germline VK A27 anda JK segment from human germline JK 5. The alignment of the 9D4 VLsequence to the germline VK A27 sequence is shown in FIG. 10. Furtheranalysis of the 9D4 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 10, and in SEQ ID NOs:16, 20 and24, respectively.

Example 3: Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit theBiological Activity of Interferon α2b

The cell line Daudi, derived from a human B-lymphoblast Burkitt'slymphoma, expresses high levels of IFNAR-1, and the growth of thesecells is inhibited by Type I interferons. To measure the functionalblocking ability of human anti-IFNAR-1 antibodies, two different assayswere performed, a cell proliferation assay and a reporter assay.

In the first assay, Daudi cells were cultured with interferon α2b in thepresence or absence of antibody and proliferation was measured by uptakeof ³[H]-thymidine. Daudi cells (ATCC CCL-213) were grown in RPMIcontaining 10% FCS, and 2 mM beta mercaptoethanol (media). Cells werespun and resuspended at a concentration of 1×10⁶ cells/ml in media withadded 1% human serum albumin (media & HS). To each well of a 96-wellplate, 100 μl of 200 U/ml interferon α2b (Schering Corporation)containing the appropriate concentration of antibody was added. 100 μlof Daudi cells in media & HS were added to the wells and the plates wereincubated for 48 hours at 37° C. The plates were pulsed with 1 μCi of³[H]-thymidine and incubated for an additional 24 hours. The plates wereharvested, collected onto a 96-well fiber filter plate, and countedusing a TopCount scintillation counter (Packard). The counts per minutewere plotted as a function of antibody concentration and the data wasanalyzed by non-linear regression, sigmoidal dose-response (variableslope) using the Prism software (San Diego, Calif.).

In the second assay, U937 cells were transfected with a construct inwhich an Interferon Stimulated Response Element was linked to a reportergene (ISRE-RG) and the ability of humanized anti-IFNAR-1 antibodies toblock IFN-induced expression of the reporter gene was measured. Thecells were grown in RPMI containing 10% FCS, and 2 mM betamercaptoethanol (media). The cells (1×10⁶ cells/ml) were resuspended inmedia with added 2% human serum. 100 μl of cells was added to a 96-wellplate. Antibodies were serially diluted in media containing 200 U/ml ofinterferon α2b (Schering corporation) and 100 μl was added to each well.The plates were incubated overnight at 37° C. Following this incubation,expression of the reporter gene was assessed by flow cytometry.Geometric mean fluorescent intensity was plotted as a function ofantibody concentration and the data was analyzed by non-linearregression, sigmoidal dose-response (variable slope) using the Prismsoftware (San Diego, Calif.).

Using the above described two assays, the potency of the 3F11 humanmonoclonal antibody was compared to the murine anti-IFNAR-1 antibody64G12 (ECACC Deposit No. 92022605) and to the humanized anti-IFNAR-1antibody D1 H3K1 (described further in U.S. Ser. No. 60/465,058). Thepotency of 3F11 showed a 5-10 fold greater potency than the mouseantibody and a 6-30 fold greater potency than the humanized antibody.The results are summarized in Table 1 below.

TABLE 1 Blocking ability of human anti-IFNAR-1 antibody on IFN alpha 2bCell Proliferation ISRE-RG Reporter Isotype (Daudi) EC₅₀ (nM) (U937)EC₅₀ (nM) 64G12 m IgG1 3.1 6.0 DI H3K1 h IgG1 9.3 8.0 3F11 h IgG1 0.31.2

Example 4: Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit theBiological Activity of IFN Omega

Using the Daudi proliferation assay described above in Example 3, theability of the human anti-IFNAR-1 antibody to inhibit IFN omegaresponses was tested. To each well of a 96-well plate, 100 μl of 200U/ml interferon omega (PBL) containing the appropriate concentration ofantibody was added. The human antibodies 3F11, 4G5, 11E2, and 9D4 were4-18 times more potent (as measured by EC₅₀) than the mouse 64G12antibody. The results are summarized in Table 2 below.

TABLE 2 Blocking ability of human anti-IFNAR-1 antibody on IFN omegaCell Proliferation Isotype (Daudi) EC₅₀ (nM) 64G12 m IgG1 5.5 DI H3K1 hIgG1 30.7 3F11 h IgG1 0.6 4G5 h IgG1 1.4 11E2 h IgG1 0.3 9D4 h IgG1 0.3

Example 5: Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit theBiological Activity of Multiple Type I IFNs

As described in Example 3, interferon alpha inhibits the proliferationof Daudi (Burkitts lymphoma, ATCC #CCL-213) cells in a dose dependantmanner. A neutralizing antibody, which blocks interferon binding to itsreceptor, will restore proliferation. Using this cell proliferationassay, the specificity of the purified human anti-IFN alpha antibodieswas examined by testing for blockade of natural lymphoblastoid IFNα,natural leukocyte interferon, 13 recombinant IFN alpha subtypes, IFNbeta and IFN omega.

Daudi cells were grown in culture medium (RPMI 1640 supplemented with10% FCS, 1×2-ME, L-glutamine and penicillin streptomycin) with andwithout the addition of IFNα in a 96 well, flat-bottomed cell cultureplate. Each type I interferon tested was assayed at EC₅₀ and mixed witha 2-fold serial titration of anti-IFNAR-1 antibody 3F11, typically from50 ug/ml (312 nM) through 381 μg/ml (2.4 pM). The antibody/IFN mixturewas added to Daudi cells in a 96-well bottomed plate to a final densityof 1×10⁴ Daudi cells/100 ul/well and incubated at 37° C., 5% CO₂, 72hrs. Proliferation was assayed with the addition of MTS (Promega), 20ul/well, and O.D. at 490 nm was measured following a further 3 hourincubation. The viable cell number was proportional to the O.D. reading.Percentage blockade of interferon was calculated relative to Daudiproliferation in the absence of IFN (=100% blockade) and in the presenceof IFN alone (=0% blockade). The 3F11 antibody was scored according tothe degree of blockade, resulting in a profile of IFNα subtypespecificity. The results demonstrated that the human anti-interferonalpha receptor 1 antibody 3F11 inhibits the action of multipleinterferon alpha subtypes, including IFNα 6, 2b, 2a, 1, 16, 10, 8, 5,14, 17, 7, 4, and 21, as well as lymphoblastoid IFN, leukocyte IFN, andIFN omega. 3F11 is a lower level inhibitor of IFN beta, althoughinhibition of greater than 50% was observed. The % blockade and standarddeviation of interferon are shown in Table 3, below.

TABLE 3 Antibody Inhibition of Multiple type I interferons 3F11 IFNBlockade (%) at 1000x Ab IFN mean sd Lymphoblastoid IFN 94.9 2.9 IFNα 6107.1 6.6 IFNα 2b 101.9 0.4 IFNα 2a 103.1 3.0 IFNα 1 111.6 1.9 LeukocyteIFN 109.4 1.4 IFNα 16 105.7 1.4 IFNα 10 96.7 5.5 IFNα 8 87.5 2.6 IFNα 5105.1 3.9 IFNα 14 100.3 1.4 IFNα 17 99.8 2.4 IFNα 7 102.8 3.2 IFNα 4100.5 2.5 IFNα 21 104.4 2.3 IFN-beta 53.0 1.7 IFN-omega 107.1 1.3

Example 6: Inhibition of IFN-Induced IP-10 Secretion by Anti-IFNAR-1Antibodies

The addition of IFN alpha 2b to cell culture media has been shown toinduce IP-10 secretion by normal peripheral blood mononuclear cells(PBMNC). The activity of human anti-IFNAR-1 antibody 3F11 was tested forinhibition of interferon induced secretion of IP-10 by normal PBMNCcultures by an ELISA binding assay.

PBMNC's were incubated in culture medium (RPMI 1640+10% FBS+1% humanserum) with leukocyte IFN, IFN alpha 2b, or IFN ω for 24-48 hours.Supernatants were collected and analyzed for IP-10/CXCL10 concentrationusing a quantitative sandwich ELISA kit (Quantikine®, R&D Systems) at a1:30 dilution according to manufacturer recommendations. The resultsdemonstrated that the human monoclonal antibody 3F11 inhibits leukocyteIFN, recombinant IFNα 2b, and recombinant IFNω induced secretion ofIP-10 by normal PBMNC culture. These results are shown in Table 4.

TABLE 4 Antibody Inhibition of IFN-Induced IP-10 Expression on NormalPBMNC Ab Treatment No IFN Leukocyte IFN IFN alpha 2b IFN omega IP-10IP-10 IP-10 IP-10 (pg/ml) (pg/ml) (pg/ml) (pg/ml) No antibody 907 26652739 2904 3F11 (5 μg/ml) 387 854 745 674 Control Ig (5 838 3512 31173960 μg/ml) * 100 U/ml of each IFN subtype was added to the cultures

Example 7: Anti-IFNAR-1 Human Monoclonal Antibodies Cross CompetitionAssay

To evaluate whether the human monoclonal antibodies bind to the sameepitope as the mouse 64G12 monoclonal antibody, a cross-competitionELISA assay was used to determine whether the antibodies competed forthe same binding epitope.

96-well plates were coated with soluble CHO-derived human IFNAR-1 at aconcentration of 1 μg/mL in freshly prepared DPBS at 100 μl/well(Mediatech). Human monoclonal antibodies 3F11, 4G5, 11E2, and 9D4 wereadded at 20 μg/mL to the wells column 1 and serially diluted at a 1:2ratio in the wells from column 1 to column 12, followed by incubationfor 45 minutes. Mouse monoclonal antibody 64G12, at an EC₇₅concentration of 0.3 μg/mL, was added at 50 μL per well and the plateswere incubated for 30 minutes. The plates were washed 3 times with Elx405 auto plate washer (BIO-TEK Instruments). A peroxidase affinitypurified F(ab′)2 goat anti-mouse IgG (Fcγ specific) antibody was diluted1:3000 in PBS and added as the detection conjugate (JacksonImmunoResearch Laboratories, cat. 115-036-0710). After a one hourincubation, the plates were washed 3 times with Elx 405 auto platewasher. An ABTS solution (800 μl ABTS stock, 8 μl 30% H₂O₂, and 100 mLcitrate phosphate buffer) at 27.8 mg/mL was added to each well andincubated for 20 minutes. The plates were read at 415 nm using 490 nm asa reference wavelength. The results are shown in FIG. 11. The resultsdemonstrate that the human anti-IFNAR-1 monoclonal antibodies, 3F11,4G5, 11E2, and 9D4 do not compete with 64G12 for binding to IFNAR-1 andthus bind to a different epitope on IFNAR-1 than 64G12.

Example 8: Antibody Inhibition of SLE Plasma Mediated Dendritic CellDevelopment

SLE plasma induces dendritic cell development from normal humanmonocytes. In this example, the purified monoclonal human anti-IFNAR-1antibody, 3F11, was tested for inhibition of dendritic cell development,as assessed by the ability of the antibodies to inhibit the induction ofthe cell surface markers CD38, MHC Class I and CD123 by SLE plasma.

A 25 ml buffy coat was diluted four fold with PBS. The sample wasseparated into 4×50 ml conical tubes, and 15 ml of lymphocyte separationmedium (ICN Biomedicals) was layered underneath. Following a 30-minutespin at 500×g, the buffy layer containing the PBMCs was removed andwashed with PBS. Cells were resuspended in culture media at 4×10⁶cells/ml. Monocytes were isolated by incubating PBMC (2.0×10⁷ cells/5ml/25 cm² flask) for 1.5 hrs at 37° C. in culture medium and thenwashing away non-adherent cells twice. Following the second wash thecells were cultured in media containing 1% heat inactivated human serum.Twenty five percent SLE patient plasma plus/minus neutralizingantibodies and isotype controls (30 ug/ml) were added to the cultureflasks; IFN alpha 2b (100 & 10 iu/ml) plus 25% normal human plasma wasused as a positive control for marker induction. Flasks were incubatedat 37° C., 5% CO₂ for three to seven days. Conditioned medium washarvested from each flask and suspension cells were recovered bycentrifugation at 1000 rpm on a Sorvall RTH-750 rotor. The pelletedcells were retained on ice and supernate was frozen at −80° C. forELISA. Adherent cells were recovered from the flask with a PBS wash (2ml), followed by 15 minute incubation in versene (3 ml), if necessary.The flask was scraped at the end of the versene incubation and the flaskwas finally rinsed with PBS wash (2 ml). Each of the PBS washes and theversene was combined with the cells recovered from conditioned mediumharvest. The pooled cell suspension was centrifuged at 1000 rpm on aSorvall RTH-750 rotor, the resulting pellet was resuspended to 300 ul instaining buffer (PBS+0.1M EDTA+2% FBS+1% HS) and dispensed 100 ul/wellinto a V-bottom 96-well plate. The plate was pulse-centrifuged at 2800rpm on a Sorvall RTH-750 rotor and pelleted cells were resuspended 25μl/well in flurochrome labeled antibodies as follows: (1) mouse anti-MHCI-FITC+mouse anti-CD38-PE, and (2) isotype controls, mouseIgG-FITC+mouse IgG-PE. The plate was incubated on ice for 45 minutes,protected from light. The cells were washed three times with theaddition of 200 ul staining buffer followed by pulse-centrifugation andfinally resuspended in 200 μl of 2% paraformaldehyde in PBS. Staining ofdendritic cells was analyzed by flow cytometry with the Becton DickinsonFACScalibur™. Gates were drawn on the Forward Scatter vs. Side Scattergraph to remove contaminating cells from the analysis. The anti-IFNAR-1human monoclonal antibody 3F11 inhibits the IFN alpha dependent processof dendritic cell development, as demonstrated by normalized expressionof cell surface markers MHC Class I, CD38, and CD123 in the presence of3F11. The results are shown below in Table 5, wherein (A) and (B)summarize results for two representative SLE donor samples.

TABLE 5 Inhibition of Dendritic Cell Maturation (A) Donor Plasma #40*(13.3 iU/mL**) MHC class I CD123 CD38 Culture Conditions MFI MFI MFI  0IFN/mL 148 14 40 10 IFN/mL 200 19 44 100 IFN/mL  229 26 63 0 206 22 473F11 115 13 32 HuIgG1 (isotype 194 22 62 control) (B) Donor Plasma #59*(75.3 iU/mL**) Culture Conditions MHC class I CD123 CD38  0 IFN/mL 22911 58 10 IFN/mL 271 12 86 100 IFN/mL  294 13 112 0 202 15 62 3F11 112 822 HuIgG1 (isotype 266 14 55 control)

Example 9: Scatchard Binding Analysis of Anti-IFNAR-1 Human Antibodiesto Daudi Cells or Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells were prepared from fresh bloodby standard protocols using Ficol separation. Daudi cells were obtainedfrom ATCC and grown in RPMI containing 10% fetal bovine serum (FBS). Thecells were washed twice with RPMI containing 10% FBS at 4 degrees andthe cells were adjusted to 4×10⁷ cells/ml in RPMI media containing 10%fetal bovine serum (binding buffer). Millipore plates (MAFB NOB) werecoated with 1% nonfat dry milk in water and stored a 4° C. overnight.The plates were washed with binding buffer and 25 ul of unlabeledantibody (1000-fold excess) in binding buffer was added to control wellsin a Millipore 96 well glass fiber filter plate (non-specific bindingNSB). Twenty-five microliters of buffer alone was added to the maximumbinding control well (total binding). Twenty-five microliters of varyingconcentration of ¹²⁵I-anti-IFNAR-1 antibody and 25 ul of Daudi cells orhuman peripheral blood mononuclear cells (4×10⁷ cells/ml) in bindingbuffer were added. The plates were incubated for 2 hours at 200 RPM on ashaker at 4° C. At the completion of the incubation the Millipore plateswere washed twice with 0.2 ml of cold binding buffer. The filters wereremoved and counted in a gamma counter. Evaluation of equilibriumbinding was performed using single site binding parameters with thePrism software (San Diego, Calif.).

Using the above Scatchard binding assay, the K_(D) of the antibody forDaudi cells and for human peripheral blood mononuclear cells wasapproximately 0.2 nM and 0.5 nM, respectively.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE 1 VH CDR1 a.a. 3F11 2 VHCDR1 a.a. 4G5 3 VH CDR1 a.a. 11E2 4 VH CDR1 a.a. 9D4 5 VH CDR2 a.a. 3F116 VH CDR2 a.a. 4G5 7 VH CDR2 a.a. 11E2 8 VH CDR2 a.a. 9D4 9 VH CDR3 a.a.3F11 10 VH CDR3 a.a. 4G5 11 VH CDR3 a.a. 11E2 12 VH CDR3 a.a. 9D4 13 VKCDR1 a.a. 3F11 14 VK CDR1 a.a. 4G5 15 VK CDR1 a.a. 11E2 16 VK CDR1 a.a.9D4 17 VK CDR2 a.a. 3F11 18 VK CDR2 a.a. 4G5 19 VK CDR2 a.a. 11E2 20 VKCDR2 a.a. 9D4 21 VK CDR3 a.a. 3F11 22 VK CDR3 a.a. 4G5 23 VK CDR3 a.a.11E2 24 VK CDR3 a.a. 9D4 25 VH a.a. 3F11 26 VH a.a. 4G5 27 VH a.a. 11E228 VH a.a. 9D4 29 VK a.a. 3F11 30 VK a.a. 4G5 31 VK a.a. 11E2 32 VK a.a.9D4 33 VH n.t. 3F11 34 VH n.t. 4G5 35 VH n.t. 11E2 36 VH n.t. 9D4 37 VKn.t. 3F11 38 VK n.t. 4G5 39 VK n.t. 11E2 40 VK n.t. 9D4 41 VH 4-34germline a.a. 42 VH 5-51 germline a.a. 43 VK L18 germline a.a. 44 VK A27germline a.a.

What is claimed is:
 1. A bispecific molecule comprising an antibody orantigen-binding portion thereof that specifically binds to humaninterferon alpha receptor 1 (IFNAR-1) comprising: (a) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;a heavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 8; a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 12; a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 16; a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:20; and a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 24; (b) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 1; a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5;a heavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 9; a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 13; a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 17; and a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:21; or (c) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 2; a heavy chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO: 6; a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 10; a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:14; a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 18; and a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 22, linked to a secondfunctional moiety having a different binding specificity than saidantibody or antigen-binding portion thereof.
 2. A composition comprisingthe bispecific molecule of claim
 1. 3. The bispecific molecule of claim1, wherein the antibody or antigen-binding portion thereof comprises aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 4; a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 8; a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 12; a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:16; a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 20; and a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:
 24. 4. A compositioncomprising the bispecific molecule of claim
 3. 5. The bispecificmolecule of claim 1, wherein the antibody or antigen-binding portionthereof comprises a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 1; a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 9;a light chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 13; a light chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 17; and a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:
 21. 6. A compositioncomprising the bispecific molecule of claim
 5. 7. The bispecificmolecule of claim 1, wherein the antibody or antigen-binding portionthereof comprises a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 2; a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 6; a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:10; a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 14; a light chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO: 18; and a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO:
 22. 8. Acomposition comprising the bispecific molecule of claim
 7. 9. Abispecific molecule comprising an antibody or antigen-binding portionthereof that specifically binds to human interferon alpha receptor 1(IFNAR-1) comprising: (a) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 32; (b) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 25 anda light chain variable region comprising the amino acid sequence of SEQID NO: 29; or (c) a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 26 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 30, linked to a secondfunctional moiety having a different binding specificity than saidantibody or antigen-binding portion thereof.
 10. A compositioncomprising the bispecific molecule of claim
 9. 11. The bispecificmolecule of claim 9, wherein the antibody or antigen-binding portionthereof comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 28 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 32. 12. A compositioncomprising the bispecific molecule of claim
 11. 13. The bispecificmolecule of claim 9, wherein the antibody or antigen-binding portionthereof comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 25 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 29. 14. A compositioncomprising the bispecific molecule of claim
 13. 15. The bispecificmolecule of claim 9, wherein the antibody or antigen-binding portionthereof comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 26 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 30. 16. A compositioncomprising the bispecific molecule of claim 15.